Different dynamics in nuclear entry of subunits of the repair/transcription factor TFIIH

We report here the different ways in which four subunits of the basal trans cription/repair factor TFIIH (XPB, XPD, p62 and p44) and the damage recogni tion XPC repair protein can enter the nucleus, We examined their nuclear lo calization by transiently expressing the gene products tagged with the enha nced green fluorescent protein (EGFP) in transfected 3T3 cells. In agreemen t with the identification of more than one putative nuclear localization si gnal (NLS) in their protein sequences, XPB, XPC, p62 and p44 chimeras were rapidly sorted to the nucleus. In contrast, the XPD-EGFP chimeras appeared mainly localized in the cytoplasm, with a minor fraction of transfectants s howing the EGFP-based fluorescence also in the nucleus. The ability of the XPD chimeras to enter the nucleus was confirmed by western blotting on frac tionated cell extracts and by functional complementation of the repair defe ct in the UV5 rodent cells, mutated in the XPD homologous gene. By deletion mutagenesis, we were unable to identify any sequence specific for nuclear localization. In particular, deletion of the putative NLS failed to affect subcellular localization and, conversely, the C-terminal part of XPD contai ning the putative NLS showed no specific nuclear accumulation. These findin gs suggest that the nuclear entry of XPD depends on its complexation with o ther proteins in the cytoplasm, possibly other components of the TFIIH comp lex.